The long-term goal of the project is to understand the structural basis of regulation of the interferon regulatory factor (IRF) family of transcription factors. IRF-3 is a family member that functions as an anti-viral switch. In uninfected cells, IRF-3 is constitutively expressed in the cytoplasm in the autoinhibited form. Upon virus infection, IRF-3 becomes phosphorylated and oligomerized, which enters the nucleus and transcriptionally activates the innate immune program through an essential interaction with the coactivator CBP/p300. The human herpesvirus 81Kaposi Sarcoma-associated herpesvirus (HHV-8/KSHV) counteracts the host's defense by expressing a viral form of IRF, vlRF-1 ,-that.interferes IRF-3 activation through sequestering CBP/p300. The proposal investigates the structural basis of IRF-3 activation by phosphorylation and CBPIp300 interaction, as well as IRF-3 inactivation by the viral gene product vlRF-I. In the preliminary studies, crystal structure of the autoinhibited IRF-3 transactivation domain was determined. Structure analysis revealed a remarkable structural homology, and potential mechanistic similarity, with the Smad family of proteins, which are transcriptional mediators of the transforming growth factor beta pathway. The structure provides a framework for investigating the mechanism of IRF-3 phospho-activation. Furthermore, diffraction quality crystals of the iRF-31CBP complex are available, which will lead to a structural determination. Three specific aims are proposed. First, the structural basis of IRF-3 phosphoactivation will be investigated to reveal the oligomeric state, oligomeric interface and structural role of phosphorylation in the activate state IRF-3. Second, the structural basis of IRF-3 interaction with CBP/p300 will be investigated to reveal the mechanism of specific recognition in transcriptional regulation. Finally, the structural basis of viral vlRF-1 competing with host IRF-3 for CBPIp300 interaction will be investigated to reveal the mechanism of competition. A combinatorial approach using X-ray crystallography, analytical ultracentrifugation, isothermal titration calorimetry, mass spectrometry, cellular biology and other biochemical techniques will be employed to investigate these questions. These studies will provide insight into the molecular mechanism of IRF signaling, providing possible targets for rational drug design to combat viral pathogenesis.